Nucleic acid detection

Abstract

This invention provides methods and kits for enriching and / or detecting a nucleic acid with at least one variant nucleotide from a nucleic acid population in a sample. Methods employ the use of enriching primers and bridge-probes for enriching and detecting target nucleic acids. Extension of the enriching primer permits amplification of the target nucleic acid having the variant nucleotide.

Description

BACKGROUND OF THE INVENTION[0001]The present invention relates to the field of detection and enrichment of a desired nucleic acid from a population of nucleic acids in a sample, especially the enrichment of rare nucleic acids containing mutations.[0002]Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. Point mutations are also usually SNPs but the term mutation is normally reserved for those SNPs with a frequency rarer than 1% and / or where there is a known correlative or functional association between the mutation and a disease (Gibson N J, 2006 Clin Chim Acta. 363(1-2):32-47). There are many applications for genotyping polymorphisms and detecting rare mutations. Rare variant detection is important for the early detection of pathological mutations, particularly in cancer. For instance, detection of cancer-associated point mutations in clinical samples can improve the identification minimal residual disease during chemotherapy and detect...

AI Technical Summary

Benefits of technology

[0010]The methods of the present invention allow for rapid, sensitive, and improved enrichment and detection of desired nucleic acids from a nucleic acid population. The improved methodology and probe also allow for rapid and sensitive detection of genetic variations in nucleic acids in samples from patients with genetic diseases or neoplasias.

[0016]In another embodiment, in step (a) the nucleic acid template forms a stem-loop structure under hybridisation conditions, wherein the double-stranded stem comprises an amplification primer binding site and the loop comprises the target nucleic acid sequence, wherein the enriching primer anneals to the diagnostic region in the loop. In step (b) the enriching primer is extended when it anneals to the diagnostic region containing a variant nucleotide; this extension opens up the-stem loop structure thereby allowing the amplification primer to anneal to the primer binding site under hybridisation conditions and promoting amplification, wherein the enriching primer is not extendable when it anneals to the diagnostic region with a normal nucleotide, whereby the stem-loop structure is intact and prevents an amplification primer from annealing to the primer binding site.

Problems solved by technology

A vast number of methods have been introduced, but no single method has been widely accepted.

Current approaches have inherent limitations due to the lack of total specificity of allele-specific primers during PCR, which creates false positives.

As a result, all current approaches have limited sensitivity and accuracy (review in Jeffreys A J and May C A, 2003 Genome Res. 13(10):2316-24).

Most mutation detection systems yield an assay signal that is difficult to validate in terms of the number of mutant molecules detected.

However, the large number of PCR reactions required, combined with background noise arising from misincorporation of nucleotides during PCR is likely to limit this approach to detection levels of about 1 variant in a population of 1000 nucleic acids.

Another limitation of many mutation detection procedures is that they replace the mutant site with a PCR primer sequence and yield short amplicons containing little, if any, information other than the presence of a putative mutant allele (review in Jeffreys A J and May C A, 2003).

The unifying problem behind all of these PCR approaches for detecting rare variants is replication infidelity during amplification.

However, this method of DNA enrichment involves multiple steps, requires large amounts of starting material and suffers from low sensitivity and efficiency.

This approach is limited, however, to the analysis of mutations at precise locations where restriction enzyme sites naturally occur.

These methods can be problematic, however.

It is difficult to find the optimal conditions for the PNA / LNA clamp; lengthy testing and redesigning are often required, and the purchase of specialised instruments may be needed.

Furthermore, PNAs are expensive and difficult to synthesise and the efficiency of inhibition is often low.

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